TLR4-dependent hepcidin expression by myeloid cells in response to bacterial pathogens.

Hepcidin is an antimicrobial peptide secreted by the liver during inflammation that plays a central role in mammalian iron homeostasis. Here we demonstrate the endogenous expression of hepcidin by macrophages and neutrophils in vitro and in vivo. These myeloid cell types produced hepcidin in response to bacterial pathogens in a toll-like receptor 4 (TLR4)-dependent fashion. Conversely, bacterial stimulation of macrophages triggered a TLR4-dependent reduction in the iron exporter ferroportin. In vivo, intraperitoneal challenge with Pseudomonas aeruginosa induced TLR4-dependent hepcidin expression and iron deposition in splenic macrophages, findings mirrored in subcutaneous infection with group A Streptococcus where hepcidin induction was further observed in neutrophils migrating to the tissue site of infection. Hepcidin expression in cultured hepatocytes or in the livers of mice infected with bacteria was independent of TLR4, suggesting the TLR4-hepcidin pathway is restricted to myeloid cell types. Our findings identify endogenous myeloid cell hepcidin production as a previously unrecognized component of the host response to bacterial pathogens.

Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections.

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops x Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel beta-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, alpha-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4-5 orders of magnitude above base-line 24-48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.

Alanylation of teichoic acids protects Staphylococcus aureus against Toll-like receptor 2-dependent host defense in a mouse tissue cage infection model.

Staphylococcus aureus is inherently resistant to cationic antimicrobial peptides because of alanylation of cell envelope teichoic acids. To test the effect of alanylated teichoic acids on virulence and host defense mediated by Toll-like receptor 2 (TLR2), wild-type (wt) S. aureus ATCC35556 (S.a.113) and its isogenic mutant expressing unalanylated teichoic acids (dlt(-)) were compared in a tissue cage infection model that used C57BL/6 wt and TLR2-deficient mice. The minimum infective doses (MID) to establish persistent infection with S.a.113 were 10(3) and 10(2) colony-forming units (cfu) in wt and TLR2(-/-) mice, respectively. The corresponding MID for dlt(-) were 5x105 and 10(3) cfu in wt and TLR2(-/-) mice, respectively. Both mouse strains showed bacterial-load-dependent inflammation with elevations in tumor necrosis factor, macrophage inflammatory protein 2, and leukocytes, with increasing proportions of dead cells. These findings indicate that alanylated teichoic acids contribute to virulence of S. aureus, and TLR2 mediates host defense, which partly targets alanylated teichoic acids.

Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge.

We report the isolation of a novel antimicrobial peptide, bass hepcidin, from the gill of hybrid striped bass, white bass (Morone chrysops) x striped bass (M. saxatilis). After the intraperitoneal injection of Micrococcus luteus and Escherichia coli, the peptide was purified from HPLC fractions with antimicrobial activity against Escherichia coli. Sequencing by Edman degradation revealed a 21-residue peptide (GCRFCCNCCPNMSGCGVCCRF) with eight putative cysteines. Molecular mass measurements of the native peptide and the reduced and alkylated peptide confirmed the sequence with four intramolecular disulfide bridges. Peptide sequence homology to human hepcidin and other predicted hepcidins, indicated that the peptide is a new member of the hepcidin family. Nucleotide sequences for cDNA and genomic DNA were determined for white bass. A predicted prepropeptide (85 amino acids) consists of three domains: a signal peptide (24 amino acids), prodomain (40 amino acids) and a mature peptide (21 amino acids). The gene has two introns and three exons. A TATA box and several consensus-binding motifs for transcription factors including C/EBP, nuclear factor-kappaB, and hepatocyte nuclear factor were found in the region upstream of the transcriptional start site. In white bass liver, hepcidin gene expression was induced 4500-fold following challenge with the fish pathogen, Streptococcus iniae, while expression levels remained low in all other tissues tested. A novel antimicrobial peptide from the gill, bass hepcidin, is predominantly expressed in the liver and highly inducible by bacterial exposure.

Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass.

We isolated a novel 22-residue, C-terminally amidated antimicrobial peptide, moronecidin, from the skin and gill of hybrid striped bass. Two isoforms, differing by only one amino acid, are derived from each parental species, white bass (Morone chrysops) and striped bass (Morone saxatilis). Molecular masses (2543 and 2571 Da), amino acid sequences (FFHHIFRGIVHVGKTIH(K/R)LVTGT), cDNA, and genomic DNA sequences were determined for each isoform. A predicted 79-residue moronecidin prepropeptide consists of three domains: a signal peptide (22 amino acids), a mature peptide (22 amino acids), and a C-terminal prodomain (35 amino acids). The synthetic, amidated white bass moronecidin exhibited broad spectrum antimicrobial activity that was retained at high salt concentration. An alpha-helical structure was confirmed by circular dichroism spectroscopy. The moronecidin gene consists of three introns and four exons. Peptide sequence and gene organization were similar to pleurocidin, an antimicrobial peptide from winter flounder. A TATA box and several consensus-binding motifs for transcription factors were found in the region 5' to the transcriptional start site. Moronecidin gene expression was detected in gill, skin, intestine, spleen, anterior kidney, and blood cells by kinetic reverse transcription (RT)-PCR. Thus, moronecidin is a new alpha-helical, broad spectrum antimicrobial peptide isolated from the skin and gills of hybrid striped bass.